The present invention relates to membranes for use in detecting the presence of an analyte.
In International patent application No W090/08783, it is disclosed how a biosensor of high sensitivity and specificity may be constructed based on a lateral segregation principle incorporating ionophores in a supported bilayer membrane. The preferred embodiment of the invention described in this application included gramicidin as the ionophore, which is known to form a conducting channel only when two monomers, one in each of the two bilayer leaflets, align themselves appropriately to form a bilayer spanning dimer. The monomers in one monolayer (called the xe2x80x9cbottomxe2x80x9d monolayer) are restrained from lateral mobility by chemical crosslinking in that monolayer, or by attachment through suitable linking groups to an underlying substrate, or by some other means. The monomers in the other (called xe2x80x9ctopxe2x80x9d) monolayer are free to diffuse laterally within that monolayer and form conducting channels by alignment with the bottom layer monomers. The top layer monomers have receptor moieties attached, which are accessible to the analyte in the solution phase above the membrane. These receptors may be any of the general types previously described, such as polyclonal or monoclonal antibodies, antibody fragments including at least one Fab fragment, antigens, etc. Another class (called xe2x80x9ccomplementaryxe2x80x9d) of receptor moieties are also attached at the membrane surface. This second class of receptor moieties is restrained from lateral mobility by attachment through to the bottom (immobilised) layer. Detection of analyte occurs when an analyte molecule is bound, at complementary sites on itself, to two receptors of both the mobile and immobilised class. This restrains the gramicidin monomer attached to one receptor from aligning itself with a monomer in the bottom layer, so causing a lowering of membrane electrical conduction which constitutes the detection event.
Such biosensors typically possess comparable surface concentrations of channel attached and immobilised receptor moieties. As such, it is necessary to ensure that all immobilised and mobile receptors are respectively of the same type, as analyte induced cross-linking between mobile channel attached receptors will typically not lead to efficient gating. In addition, the detection sensitivity of such a device in a convenient time (approximately 100 seconds) is set by the known diffusion rate constant, Kon (approximately 108 Mxe2x88x921s1) for binding from solution under physiological conditions. In order that a significant (approximately 50%) fraction of detection sites be occupied (here channels to be gated), the analyte concentration, c, must satisfy,
c greater than 1/(KonX 100)xe2x80x83xe2x80x83(1)
This general requirement limits any detection device, operating under the above requirements, without some additional means of detection amplification and sets an analyte detection concentration limit of approximately 10xe2x88x9210M.
The present inventors have found that an improvement in sensitivity of membranes for use in detecting the presence of an analyte can be obtained by increasing the ratio of fixed receptor molecules to mobile receptor molecules above a ratio of 1:1.
Accordingly, the present invention consists in a membrane for use in detecting the presence of an analyte, the membrane comprising an array of closely packed self-assembling amphiphilic molecules and a plurality of first and second receptor molecules, the first receptor molecules being reactive with one site on the analyte and second receptor molecules being reactive with another site on the analyte, the first receptor molecules being prevented from lateral diffusion within the membrane whilst the second receptor molecules are free to diffuse laterally within the membrane, the membrane being characterized in that the ratio of first receptor molecules to second receptor molecules is 10:1 or greater.
In a preferred embodiment of the present invention the ratio of first receptor molecules to second receptor molecules is in the range 10:1 to 105:1 and is preferably about 1,000:1.
In yet a further preferred embodiment of the present invention the first and second receptor molecules bind to different epitopes on the analyte.
In a preferred embodiment of the present invention a membrane is a bilayer and includes a plurality of ionophores comprising first half membrane spanning monomers provided in one layer and second half membrane spanning monomers provided in the other layer, the first half membrane spanning monomers being prevented from lateral diffusion within the membrane whilst the second half membrane spanning monomers are free to diffuse laterally within the membrane, the second receptor molecules being bound to the second half membrane spanning monomers such that the binding of the analyte to the first and second receptor molecules causes a change in the conductance of the membrane.
The first and second half membrane spanning monomers may be any such molecules known in the art, however, it is presently preferred that the first and second half membrane spanning monomers are gramicidin or one of its derivatives.
In a further preferred embodiment of the present invention the membrane includes membrane spanning lipids. It is further preferred that the first receptor molecules are attached to the membrane spanning lipids.
The present inventors have also developed a novel method of increasing the number of first receptor molecules by using a loose polymer network attached to the membrane. Accordingly, in another embodiment of the present invention linear polymer chains of radius of gyration of approximately 100 to 300 xc3x85 are attached to the surface of the membrane, the first receptor molecules being attached to the linear polymer chains.
The linear polymer chains are preferably attached to the membrane at one or two points through suitably functionalised lipids in the top layer. These may be membrane spanning lipids.
In a preferred embodiment of the present invention the radius of gyration of the linear polymer chains is approximately 200 xc3x85. All antibodies then attached to the polymer chain will be within approximately 500 xc3x85 of the surface of the membrane.
It is preferred that the ratio of linear polymer chains to lipids in the membrane is approximately 1:104. This should give xe2x80x9cloose contactxe2x80x9d packing of the polymer on the surface of the membrane thereby allowing free diffusion of the first half membrane spanning monomers.
The polymer chains are preferably condensed polyethylene glycol.
The radius of gyration, So is given by
S02=⅓xcex12l2n for a chain containing tetrahedral bonds of length, l, n is the number of links and xcex12 a constant characteristic or the polymer. For PEO (polyethylene oxide) (xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94) m 1 is the average CH2xe2x80x94CH2 or CH2xe2x80x940 bond length, xcx9c1.5 xc3x85, and xcex12xcx9c2.
n=3 m for PEO (ie xcx9c3 times no. of monomer units).
If Soxcx9c200 xc3x85, then nxcx9c25,000 (mwxcx9c400,000).
The mean mass fraction of polymer in the 500 xc3x85 thick layer is       ∼                  4        xc3x97                  10          5                            6        xc3x97                  10          23                xc3x97                              (                          5              xc3x97                              10                                  -                  6                                                      )                    3                      ∼    0.005    ,
i.e.  less than 1%.
This should still permit reasonably easy lateral movement of antibody/ion channel complexes on the surface.
The readily available form of PEO is PEG, poly-ethyleneglycol. OHxe2x80x94CH2xe2x80x94CH2xe2x80x94(CH2xe2x80x94CH2xe2x80x94O) mCH2xe2x80x94CH2xe2x80x94OH. This has hydroxyl groups at each chain end. So a chain with nxcx9c25,000 and xcx9c10 functional attachment points for membrane anchoring or antibody binding) might be formed by condensing shorter chained PEG (nxcx9c2,500) with a suitably bifunctional (e.g. dicarboxylic acid) molecule containing also a side chain (e.g. hydrazide) for antibody/lipid attachment.
It is envisaged that a common attachment chemistry be used for the antibodies and membrane attachment lipids (e.g. hydrazide linkage to aldehydes). The polymer may be attached first to the membrane surface (by adding it as a xcx9c1% solution in saline) and the unreacted excess removed. Then suitably activated antibody would be added and reacted.
This method of attachment has several potential advantages over that proposed earlier for anchoring proteins directly at the surface through short linkages to membrane spanning lipids.
1) The antibodies now have much greater local freedom to orient for a cross-linking reaction. This should approximate the conditions obtained within an ELISA assay. If the inner layer tethered gramicidin density is no higher than xcx9c1:104, the statistical gating off of each on analyte induced cross-linking of channel and polymer bound antibodies should still apply. Even with single point attachment of the polymer chains to mobile top layer lipids, the weakly overlapping mass of polymer xe2x80x9cspheresxe2x80x9d should resist lateral mobility on the hundreds of xc3x85 or greater length scale.
2) A substantially higher surface density of xe2x80x9cimmobilisedxe2x80x9d antibodies is possible.
3) The loose, xe2x80x9cbio compatiblexe2x80x9d polymer net at the surface will probably reduce non-specific protein binding to the membrane.
In a further preferred embodiment of the present invention the membrane is attached to an electrode via linking molecules such that a space exists between the membrane and the electrode. Preferred linking molecules are those disclosed in application No PCT/AU92/00132 and PCT/AU93/00509. The disclosure of each of these applications is incorporated herein by reference.
The first half membrane spanning monomers may be prevented from diffusing laterally in the membrane using any of a number of known techniques, however, it is presently preferred that the first half membrane spanning monomers are attached to the electrode via linker groups.
In yet a further preferred embodiment of the present invention a fluorescent quencher is attached to the first receptor molecule and a fluorescent species is attached to the second receptor molecule.
In such an arrangement the membrane is illuminated by the exciting wavelength of the fluorescent species. Upon addition of the analyte, the analyte is bound to the receptors. Due to the greater proportion of bound first receptor it is more probable that the analyte will be bound to the first receptor molecule. The mobile receptor diffusing through the membrane will then come into contact with the analyte bound to the first receptor molecule. The second receptor molecule will then bind to the analyte and the fluorescent group will be quenched and the emitted fluorescence will drop. Using this approach the presence of an analyte can be detected by a drop in fluorescence.
As used herein the term xe2x80x9creceptor moleculexe2x80x9d is used in its widest context. The receptor molecule may be any chemical entity capable of binding to the desired analyte. The receptor molecule is any compound or composition capable of recognizing another molecule. Natural receptors include antibodies, enzymes, lectins, dyes and the like. For example, the receptor for an antigen is an antibody while the receptor for an antibody is either an anti-antibody or preferably, the antigen recognized by that particular antibody.
The first and second receptor molecules may be the same or different and are preferably selected from the group consisting of polyclonal or monoclonal antibodies, fragments thereof including at least one Fab fragment, antigens, lectins, haptens and dyes. It is most preferred that the receptor molecules are antibodies or fragments thereof.
It will be clear to persons skilled in the art that the membrane of the present invention may advantageously incorporate a number of lipid and linker compounds described in PCT/AU93/00509. It is intended that such modifications are within the scope of the present invention and the disclosure of this co-pending application is incorporated herein by reference.